Biology subjects

Dunn, D. E.

Publications and source records attributed to Dunn, D. E..

3 recordsLinked to original sources

bioRxiv2025

Evidence for Functional Regulation of the KLHL3/WNK Pathway by O-GlcNAcylation

The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked {beta}-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.

cell biology↗

bioRxiv2025

Targeting the ferroptosis pathway: A novel compound, AZD1390, protects the brain after ischemic stroke

BackgroundFerroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation. This process has been implicated in various diseases, including ischemic stroke. Ischemic stroke leads to oxidative stress, iron overload, and reactive oxygen species (ROS) accumulation, which collectively may trigger ferroptotic neuronal cell death. However, the regulatory mechanisms of ferroptosis in stroke remain poorly understood. Previous studies have identified ataxia telangiectasia mutated (ATM), a DNA damage kinase, as a critical regulator of ferroptosis. However, the therapeutic potential of this discovery remains unknown. MethodsWe investigated the effect of ATM inhibitors, including the brain-penetrant AZD1390, on ferroptosis using in vitro, ex vivo, and in vivo models of ischemic stroke. Our analysis included assessments of cell viability, lipid peroxidation, ferroptosis marker expression, and infarct volume. ResultATM inhibitors significantly alleviated ferroptosis-induced cell death in cultured cells and ex vivo murine brain slice cultures. In the oxygen-glucose deprivation (OGD) stroke model, treatment with AZD1390 reduced the expression of ferroptosis markers (xCT and PTGS2) and diminished neuronal cell death in rat and mouse brain slices. Furthermore, in a mouse model of ischemic stroke, AZD1390 decreased infarct volume confirming its therapeutic efficacy in vivo. ConclusionsThis study identifies ferroptosis as a critical mechanism in ischemic stroke-induced neuronal cell death and highlights ATM inhibition, particularly with AZD1390, as a promising therapeutic candidate for mitigating stroke-associated damage. Targeting ferroptosis may provide a translationally relevant strategy to mitigate neuronal injury and improve clinical outcomes for stroke patients.

neuroscience↗

bioRxiv2024

Protein CoAlation on TXNRD2 regulates mitochondrial thioredoxin system to protect against ferroptosis

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

cell biology↗